1. Field of the Invention
The measurement of plasma renin activity (PRA) is considered of primary importance in the investigation of hypertensive conditions. For example, it is widely employed to determine whether hypertension is due to primary aldosteronism or secondary aldosteronism. Both conditions reveal a high aldosterone secretion rate; primary aldosteronism occuring with a low renin activity, secondary aldosteronism exhibiting a high renin activity. It is, then, important to distinguish between low renin values and the lower part of the normal spectrum. In other words, conditions under which plasma renin activity are measured must be maximized to differentiate truly low renin values from the low but normal values. Most commercial kits on the market that determine Angiotensin I as a measure of PRA have neglected this consideration and, as a result, cannot differentiate between truly low renin values and low but normal values.
Plasma renin activity (PRA) is generally measured by the quantitative determination of Angiotensin I. Angiotensin I can be lost after it has been produced if it is not effectively inhibited as can be seen from the multireaction system shown below.
______________________________________ (1) Angiotensinogen Renin Angiotensin I .fwdarw. (2) Angiotensin I Converting enzyme Angiotensin II .rarw. .fwdarw. Ca++, Mg++ + Dipeptide (3) Angiotensin II Angiotensinase Inactive Peptides .fwdarw. ______________________________________
It is of fundamental importance then to have one or more compounds present in this system, which will serve as inhibitors of the side reactions and will therefore prevent the loss of Angiotensin I.
In addition, the measurement of Angiotensin I is now assuming importance in the study of platelet-dependent thrombotic phenomena. A stimulation of immunoreactive material resembling prostaglandin E by Angiotensin II has been reported. Prostaglandin secretion by endothelium appears to excercise a significant role in platelet-dependent thrombotic phenomena and in local control of vascular permeability as reported by Gimbrone and Alexander. Angiotensin II may be directly monitored by its precursors Angiotensin I. Obviously, an accurate and rapid method will meet a presently growing need for Angiotensin I monitoring in the clinical patient.
2. Description of the Prior Art
I have found in my application Ser. No. 419,628, filed Nov. 28, 1973, that using phenylmethyl sulfonylfluoride (PMSF) at a final concentration of 1.32 mg/ml plasma and adjusting the pH of the plasma to pH 6.0 the yield of Angiotensin I measured is optimized when compared with Hydroxyquinoline and Dimercaprol or with Diisopropylfluorophosphate (DFP) for which clinically normal values have been established. In this method the addition of PMSF was such as to make the sample dilution negligible.
In addition, commercially available kits for the measurement of plasma renin activity (PRA) by determination of Angiotensin I use an incubation period of 24 .+-. 2 hours at 4.degree. C, after the addition of antibody using charcoal to separate the free from the antibody bound specie in the radioimmunoassay. This long incubation at 4.degree. C makes it impossible to obtain results in the laboratory the same day that the sample arrives. Also, the use of charcoal to separate the free from the antibody bound specie requires the step of carefully removing each supernatant from the pellet after centrifugation as charcoal pellets are easily disturbed. Furthermore, as charcoal is a non-specific adsorbant, the time elapsed between the addition of charcoal and the centrifugation of the tubes is rather critical creating a real possible source of error, if many samples are being processed.
Reports have appeared in the literature, Clinical Chemistry, by M. James Barrett and Patricia S. Cohen, Vol. 18, pages 1339-1342, 1972, suggesting the use of polyethylene glycol in sodium barbital buffer (25 mmol/liter, pH 8.6) to separate free from antibody bound specie after incubation of 24 .+-. 2 hours at 4.degree. C using inhibitors which do not yield optimal amounts of Angiotenin I. This technique also makes it impossible to assay the sample the same day that it is received in the laboratory.
The different methods can be summarized in the chart, which follows:
__________________________________________________________________________ Present Method: __________________________________________________________________________ Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Addition of Addition of Add 100 - inhibitor label and 250 .mu.l which opti- addition of serum and mizes yield antibody carbowax of Angio- tensin I (PMSF) .dwnarw. .dwnarw. Sample Incubate Incubate Centrifugation Count Taken .dwnarw. at 37.degree. C 11/2 hr Pellet at 28.degree. C .fwdarw. .fwdarw. .fwdarw. .fwdarw. Other Methods: __________________________________________________________________________ Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7 Step 8 Addition of Addition of Addition of inhibitors label and a) charcoal or other than addition of add 250 .mu.l PMSF antibody b) plasma and carbowax (see Clinical Chemistry .dwnarw. .dwnarw. article) Sample Incubate Incubate Centrifugation Count Taken at 37.degree. C 24 .+-. 2 hrs .dwnarw. Super with at 4.degree. C Charcoal .fwdarw. .fwdarw. .fwdarw. .fwdarw. or Pellet with Carbowax __________________________________________________________________________
In the above chart, the steps within the dotted circles in the presence of PMSF as inhibitor provide the same quantitative results. These steps are after addition of label and antibody so that the method is specific as a part of the radioimmunoassay and in no other methodology.
The advantages are as follows:
1. time of second incubation is reduced from 24 .+-. 2 hours to 1 to 2 hours, allowing the test to be done the same day;
2. time dependent errors are avoided with polyethylene glycol instead of charcoal, as charcoal is a non-specific adsorbant the time element after addition of charcoal and before centrifugation (step 7) is critical. In other words, if one uses charcoal and doesn't centrifuge immediately, it starts adsorbing what it should not, see article in Clinical Chemistry by Barret and Cohen; and,
3. technician's time involved in the separation of the free from the bound specie after centrifugation is substantially shortened and errors are avoided in this separation step.
As the pellet formed after centrifugation when using polyethylene glycol is not easily disturbed, and it is this pellet in advantage 3, above, that one counts, all supernatants can be discarded in one operation by simply inverting the rack of tubes (when tubes are firmly held by the rack). The pellets remaining firmly attached to the bottom of the tubes. By contrast, the careful separation of the supernatant, which one counts in the case of charcoal, from the easily disturbed charcoal pellet (after centrifugation) requires much more careful handling of each individual tube and demands more of the technician's labor. This labor can be considerable if the number of samples being processed is large. In addition, a possible error in this step is introduced unless careful separation is achieved.